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Published May 2020 | Supplemental Material
Journal Article Open

Spatially distinct physiology of Bacteroides fragilis within the proximal colon of gnotobiotic mice


A complex microbiota inhabits various microenvironments of the gut, with some symbiotic bacteria having evolved traits to invade the epithelial mucus layer and reside deep within the intestinal tissue of animals. Whether these distinct bacterial communities across gut biogeographies exhibit divergent behaviours is largely unknown. Global transcriptomic analysis to investigate microbial physiology in specific mucosal niches has been hampered technically by an overabundance of host RNA. Here, we employed hybrid selection RNA sequencing (hsRNA-Seq) to enable detailed spatial transcriptomic profiling of a prominent human commensal as it colonizes the colonic lumen, mucus or epithelial tissue of mice. Compared to conventional RNA-Seq, hsRNA-Seq increased reads mapping to the Bacteroides fragilis genome by 48- and 154-fold in mucus and tissue, respectively, allowing for high-fidelity comparisons across biogeographic sites. Near the epithelium, B. fragilis upregulated numerous genes involved in protein synthesis, indicating that bacteria inhabiting the mucosal niche are metabolically active. Further, a specific sulfatase (BF3086) and glycosyl hydrolase (BF3134) were highly induced in mucus and tissue compared to bacteria in the lumen. In-frame deletion of these genes impaired in vitro growth on mucus as a carbon source, as well as mucosal colonization of mice. Mutants in either B. fragilis gene displayed a fitness defect in competing for colonization against bacterial challenge, revealing the importance of site-specific gene expression for robust host-microbial symbiosis. As a versatile tool, hsRNA-Seq can be deployed to explore the in vivo spatial physiology of numerous bacterial pathogens or commensals.

Additional Information

© 2020 Springer Nature Limited. Received 25 July 2018; Accepted 03 February 2020; Published 09 March 2020. We thank E. Hsiao, E. Martens, D. Gevers, C. Desjardins, B. Haas and J. Livny for helpful discussions, and members of the Mazmanian laboratory for comments. G.P.D. was supported by an NIH training grant no. 5T32 GM07616, National Science Foundation Graduate Research Fellowship no. DGE-1144469 and the Center for Environmental Microbial Interactions at Caltech. The project was funded by NIH grant no. U19AI110818 to the Broad Institute; NIH grant no. DK110534 to H.C.; NIH grant nos. GM099535 and DK078938, and the Heritage Medical Research Institute to S.K.M. Data availability: RNA-Seq and hsRNA-Seq data have been deposited in the NCBI SRA under accession no. PRJNA438372. The B. fragilis NCTC 9343 genome used for mapping is available at GenBank under assembly no. GCA_000025985.1. Source data for Figs. 1–4 and Source Data Extended Data Figs. 1, 8, 9 and 10 are included in this article. Code availability: The code used in the analysis is available at https://github.com/wenchichou/bugInHost. Author Contributions: G.P.D. and S.K.M. conceived the study. G.P.D., W.-C.C., G.G., A.M.E. and S.K.M. designed the study. G.P.D. prepared the samples for sequencing and performed the mouse colonization and microbiology experiments. D.C., P.R., J.B., A.M. and G.G. performed the hybrid capture and sequencing experiments. W.-C.C., A.L.M. and T.A. performed the computational analysis. H.C. performed the colitis model and flow cytometry. P.B.E. scored the sections for histology. G.P.D., W.-C.C. and A.L.M. created the figures. A.M.E. and S.K.M. supervised the work. G.P.D., W.-C.C., A.L.M., A.M.E. and S.K.M. wrote the paper. All authors provided input on the paper. The authors declare no competing interests.

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August 22, 2023
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